In the 1980's, it was discovered by researchers that the endothelium tissue of the human body produced nitric oxide (NO), and that nitric oxide is an endogenous vasodilator, namely, an agent that widens the internal diameter of blood vessels. Prior to the 1980's, nitric oxide was most commonly known as an environmental pollutant that was produced as a byproduct of combustion. At high concentrations, inhaled nitric oxide is toxic to humans. At low concentrations, researchers have discovered that inhaled nitric oxide can be used to treat various pulmonary diseases in subjects. For example, nitric oxide has been investigated for the treatment of subjects with increased airway resistance as a result of emphysema, chronic bronchitis, asthma, adult respiratory distress syndrome (ARDS), and chronic obstructive pulmonary disease (COPD).
NO has been shown to play a critical role in various bodily functions, including the vasodilatation of smooth muscle, neurotransmission, regulation of wound healing and immune responses to infection such as bactericidal action directed toward various organisms (Moncada et al., 1991, Pharmacol Rev, 43: 109-42; De Groote et al., 1995, Clin Infect Dis, 21(suppl 2): S162-164). NO has been demonstrated to play an important role in wound healing through vasodilatation, angiogenesis, anti-inflammatory and antimicrobial action (Witte et al., 2002, Amer J of Surg, 183: 406-12). NO is a lipophilic signaling molecule with a small stokes radius that enables it to readily cross the plasma membrane into the cytosol. It is hypothesized that the antimicrobial and cellular messenger regulatory properties of this molecule, delivered in an exogenous gaseous form, might easily enter the wound milieu and be useful in optimizing the healing of chronic wounds with specific actions directed at reducing bacterial burden, reducing exudate and improving endogenous debridement.
The therapeutic potential of nitric oxide donors for cutaneous lesions, as a broad-spectrum antimicrobial seems promising (Fang, 1997, Amer Soc Clin Invest, 33: 2818-25; Vazquez-Torres et al., 1999, Nitric Oxide and Infection, 475-88). However, to date, this approach has not been realized in clinical application. This may be due to the toxic side effects of the carrier compounds of solid, liquid, cream, or other non-gaseous NO donors and the acidic environment required for release of the NO molecule (Omerod et al., 1999, J Invest Dermatol, 113: 392-7; Bauer et al., 1998, Wound Repair Regen, 6: 569-77). Endogenous approaches such as intracellular nitric oxide synthase (NOS) stimulation and wound dressings with either NO-donors or saturated NO-containing solutions have also failed to release consistent steady-state concentrations of NO (Shabini et al., 1996, Wound Repair Regen, 4: 353-63). Benjamin, and colleagues, describes a system using inorganic nitrite and an organic acid to produce NO on the skin surface (Weller et al., 1998, J Am Acad Dermatol, 38: 559-63). However, they describe the system as messy, impractical, causing pain in open wounds and possibly causing further damage to wounds. Recently, Hardwick, et al., refined the system using a selectively permeable membrane between the reactants and the wound. They reported that in an in vitro model it was effective at reducing microbial load (Hardwick et al., 2011, Clinical Sci, 100: 395-400).
It has been postulated that NO may be involved in wound macrophage infiltration regulation by down regulating cytokine-induced RANTES expression (Frank et al., 2000, Biochem J, 347 Pt 1: 265-73). NO may also reduce inflammation by its ability to scavenge reactive oxygen species (Bandarage et al., 2001, Mini Rev Med Chem, 1: 57-70; Patel et al., 1999, Biochim Biophys Acta, 1411: 385-400). While the inflammatory response is integral to wound healing, an aberrant inflammatory response is believed to be one causal factor in chronic wounds. NO inhibits platelet aggregation, assists in maintaining vascular tone, and inhibits mast cell degranulation (Hickey, 2001, Clin Sci, 100:1-12; Delledonne et al., 2003, Antioxid Redox Signal, 5: 33-41). NO produced constitutively by endothelial cells has been shown to have an on-going anti-inflammatory. This may in part be due to its effect on platelet aggregation. Inducible nitric oxide synthase (iNOS) is upregulated during the inflammatory response. Studies have shown that iNOS derived NO may also have anti-inflammatory characteristics effect (Hickey, 2001, Clin Sci, 100:1-12). Collectively, by maintaining vascular tone, promoting angiogenesis, moderating inflammation and inhibiting mast cell degranulation, NO can be viewed as an important molecule for exudate management. These actions of NO should nearly eliminate all but a “normal” level of exudate production.
Until recently, use of any oxide of nitrogen in the human model had not been contemplated. The discovery that NO is a significant messenger molecule in the cardiovascular system and capable of acting as a potent and specific pulmonary vasodilator resulted in the gaseous form of NO (gNO) being approved as an inhaled drug in the USA for the treatment of pulmonary hypertension of the newborn in 1999 (Miller, 2003, Journal for Respiratory Care Practitioners, 10: 10-12). There is now more than a decade of experience with the safe delivery, monitoring and understanding of NO in the clinical environment for vascular uses. With the experience of safe use and a better understanding of nitric oxide pharmacokinetics, interest has developed to closely examine the potential bacteriocidal effects of directly applied gaseous NO. Bactericidal effects of NO have been studied as far back as 1941 in the meat processing industry (Tarr, 1941, Nature, 147: 417-18). These original studies resulted in the use of nitrites in a mildly acidic environment forming nitrous acid, which dismutates to NO (Shank et al., 1962, Appl Bicrobiol, 10: 185-9).
Endogenous NO clearly plays a crucial role as an antimicrobial mediator in the human body. Although initially controversial, it is now well established that human macrophages generate NO as a primary mechanism of killing foreign microbes (MacMicking et al., 1997, Ann Rev Immunol, 15: 323-50). In both animal models and humans there is data that supports the concept that the inducible form of nitric oxide synthase (iNOS or NOS2) can be up-regulated by cytokines and bacterial products like lipopolysaccharide and lipoteichoic acid that are related to the body's response to infection (MacMicking et al., 1997, Ann Rev Immunol, 15: 323-50; Hibbs et al., 1992, J Clin Invest, 89: 867-77). Systemic nitrite and nitrate (end products of NO metabolism) levels are often elevated during infection (Ochoa et al., 1991, Ann Surg, 214: 621-6). It has also been reported that there is an upregulation of NOS or production of NO directly at the site of infection (Nicholson et al., 1996, J Exp Med, 183: 2293-2302; Stengler et al, 1996, J Exp med, 183: 1501-14).
Evidence that nitric oxide may play a role in the host defense mechanism is provided during exacerbation of infection through inhibition of nitric oxide synthases. NOS inhibitors are relatively selective, nontoxic compounds that inhibit this class of enzymes and can be administered to cultured cells, experimental animals, and people. The NOS inhibitors have been shown to worsen the course of diseases caused by an impressive array of phyla—viruses, bacteria, fungi, protozoa, and helminthes (Shank et al., 1962, Appl Microbiol, 10: 185-9). It has been reported that NOS expression is associated with good clinical outcomes in individuals infected with malaria (Anstay et al., 1999, Nitric Oxide and Infection). There are many studies that support enhancement of microbial proliferation by NOS inhibition in phagocytes and killing or inhibition of microbes by NO— donor compounds (DeGroote et al., Nitric Oxide and Infection).
Nitric oxide has also been investigated for its use as a sterilizing agent. It has been discovered that nitric oxide will interfere with or kill the growth of bacteria grown in vitro. PCT International Application No. PCT/CA99/01123, published Jun. 2, 2000, discloses a method and apparatus for the treatment of respiratory infections by nitric oxide inhalation. Nitric oxide has been found to have either an inhibitory and/or a cidal effect on pathogenic organisms.
While nitric oxide has shown promise with respect to certain medical applications, delivery methods and devices must cope with certain problems inherent with gaseous nitric oxide delivery. For example, exposure to very high concentrations of inhaled nitric oxide is toxic. Even lower levels of inhaled nitric oxide, however, can be harmful if the time of exposure is relatively high. For example, the Occupational Safety and Health Administration (OSHA) has set exposure limits for inhaled nitric oxide in the workplace at 25 ppm time-weighted averaged for eight (8) hours. It is extremely important that any device or system for delivering nitric oxide include features that diminish the leaking of significant amounts of nitric oxide into poorly ventilated spaces of the surrounding environment. If the device is used within a closed space, such as a hospital room or at home, dangerously high levels of nitric oxide can build up in a short period of time.
Another potential problem with using nitric oxide is that nitric oxide oxidizes in the presence of oxygen to form nitric dioxide, which when inhaled is toxic, even at levels lower than those of nitric oxide. If compensatory precautions are not taken, unacceptably high levels of nitric dioxide can develop, especially in closed, unventilated spaces. The rate of oxidation of nitric oxide to nitric dioxide is dependent on numerous factors, including the concentration of nitric oxide, the concentration of oxygen, and the time available for reaction. For example, in a mixture of 1000 ppm nitric oxide and 21% oxygen, it takes about 3.5 minutes for half of the nitric oxide to react to become nitric dioxide (Chironna and Altshuler, 1999, Pollution Engineering p. 33-36). Since nitric oxide will react with the oxygen in the air to convert to nitric dioxide, it is desirable to minimize incidental contact between the nitric oxide gas and the outside environment.
Another limitation of gaseous NO delivery to a subject is that the subject's skin provides a substantial barrier for effective delivery of NO to sites below the skin surface. This has restricted the use of gaseous NO to surface treatments. The skin barrier has thus prevented gaseous NO-based therapies at sites underneath the skin and within the body. Thus, there may be more clinical benefit for gaseous NO if it can be delivered below the outermost layer of the skin.
There remains a need for a device and method for the delivery of nitric oxide to treatment sites at the skin surface and below the skin surface. The present invention fulfills this need.